Immunogenic compositions comprising liver stage malarial antigens

ABSTRACT

A vaccine composition comprising a Th1-inducing adjuvant in combination with a protecting Liver Stage Antigen or immunological fragment thereof of a human malaria parasite, especially  Plasmodium falciparum , with the proviso that when the immunological fragment is an immunological fragment of LSA-3 the Th1-inducing adjuvant is not Montanide. In one preferred aspect the Th1-inducing adjuvant comprises QS21, De-O-acylated monophosphoryl lipid A (3D-MPL) and an oil in water emulsion wherein the oil in water emulsion has the following composition: a metabolisible oil, such a squalene, alpha tocopherol and tween 80. In a further preferred aspect the protecting Liver Stage Antigen is Liver Stage Antigen 3 (LSA-3) or an immunological fragment thereof. A multivalent vaccine composition is also provided comprising the vaccine composition of the invention and in addition at least one other protecting antigen or an immunological fragment thereof, of a malaria parasite.

[0001] The present invention relates to novel vaccine formulations, tomethods of their production and to their use in medicine. In particular,the present invention relates to a malaria antigen known as Liver StageAntigen 3, or an immunological fragment thereof, in association with aTh-1 inducing adjuvant such as an oil in water emulsion or a vesicularadjuvant formulation comprising cholesterol, a saponin and optionally alipopolysaccharide derivative. These and other aspects of the inventionare described hereinbelow.

[0002] It has long been known that enterobacterial lipopolysaccharide(LPS) is a potent stimulator of the immune system, although its use inadjuvants has been curtailed by its toxic effects. A non-toxicderivative of LPS, monophosphoryl lipid A (MPL), produced by removal ofthe core carbohydrate group and the phosphate from the reducing-endglucosarnine, has been described by Ribi et al (1986, Immunology andImmunopharmacology of bacterial endotoxins, Plenum Publ. Corp., NY,p407-419).

[0003] A farther detoxified version of MPL results from the removal ofthe acyl chain from the 3-position of the disaccharide backbone, and iscalled 3-O-Deacylated monophosphoryl lipid A (3D-MPL). 3 De-O-acylatedmonophosphoryl lipid A is known from GB2 220 211 (Ribi). Chemically itis a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6acylated chains and is manufactured by Ribi Immunochem Montana. GB2122204B also discloses the preparation of diphosphoryl lipid A, and3-O-deacylated variants thereof. Other purified and syntheticlipopolysaccharides have been described (U.S. Pat. No. 6,005,099 and EP0 729 473 B1; Hilgers et al., 1986, Int.Arch.Allergy.Immunol.,79(4):392-6; Hilgers et al., 1987, Immunology, 60(1):141-6; and EP 0 549074 B1).

[0004] A preferred form of 3 De-O-acylated monophosphoryl lipid A(3D-MPL) is in the form of an emulsion having a small particle size lessthan 0.2 μm in diameter, disclosed in International Patent ApplicationNo. WO 92/116556 (SmithKline Beecham Biologicals s.a.). See also WO94/21292.

[0005] Aqueous formulations comprising monophosphoryl lipid A and asurfactant have been described in WO98/43670A2.

[0006] Saponins are taught in: Lacaille-Dubois, M and Wagner H. (1996. Areview of the biological and pharmacological activities of saponins.Phytomedicine vol 2 pp 363-386). Saponins are steroid or triterpeneglycosides widely distributed in the plant and marine animal kingdoms.Saponins are noted for forming colloidal solutions in water which foamon shaking, and for precipitating cholesterol. When saponins are nearcell membranes they create pore-like structures in the membrane whichcause the membrane to burst. Haemolysis of erythrocytes is an example ofthis phenomenon, which is a property of certain, but not all, saponins.

[0007] Saponins are known as adjuvants in vaccines for systemicadministration. The adjuvant and haemholytic activity of individualsaponins has been extensively studied in the art (Lacaille-Dubois andWagner, supra). For example, Quil A (derived from the bark of the SouthAmerican tree Quillaja Saponaria Molina), and fractions thereof, aredescribed in U.S. Pat. No. 5,057,540 and “Saponins as vaccineadjuvants”, Kensil, C. R., Crit Rev Ther Drug Carrier Syst, 1996, 12(1-2):1-55; and EP 0 362 279 B1. Particulate structures, termed ImmuneStimulating Complexes (ISCOMS), comprising fractions of Quil A arehaemolytic and have been used in the manufacture of vaccines (Morein,B., EP 0 109 942 B1; WO 96/11711; WO 96/33739). The haemolytic saponinsQS21 and QS17 (HPLC purified fractions of Quil A) have been described aspotent systemic adjuvants, and the method of theirproduction isdisclosed in U.S. Pat. No. 5,057,540 and EP 0 362 279 B 1. Othersaponins which have been used in systemic vaccination studies includethose derived from other plant species such as Gypsophila and Saponaria(Bomford et al., Vaccine, 10(9):572-577, 1992).

[0008] QS21 is a Hplc purified non toxic fraction of a saponin from thebark of the South American tree Quillaja Saponaria Molina and its methodof its production is disclosed (as QA21) in U.S. Pat. No. 5,057,540.

[0009] Oil emulsion adjuvants have been known for many years, includingwork on Freund's complete and incomplete mineral oil emulsion adjuvants.Since that time much work has been performed to design stable and welltolerated alternatives to these potent, but reactogenic, adjuvantformulations.

[0010] Many single or multiphase emulsion systems have been described.Oil in water emulsion adjuvants per se have been suggested to be usefulas adjuvant compositions (EP 0 399 843B), also combinations of oil inwater emulsions and other active agents have been described as adjuvantsfor vaccines (WO 95/17210). Other oil emulsion adjuvants have beendescribed, such as water in oil emulsions (U.S. Pat. No. 5,422,109; EP 0480 982 B2) and water in oil in water emulsions (U.S. Pat. No.5,424,067; EP 0 480 981 B).

[0011] In order for any oil in water composition to be suitable forhuman administration, the oil phase of the emulsion system preferablycomprises a metabolisable oil. The meaning of the term metabolisable oilis well known in the art. Metabolisable can be defined as “being capableof being transformed by metabolism” (Dorland's Illustrated MedicalDictionary, W. B. Sanders Company, 25th edition (1974)). The oil may beany vegetable oil, fish oil, animal oil or synthetic oil, which is nottoxic to the recipient and is capable of being transformed bymetabolism. Nuts (such as peanut oil), seeds, and grains are commonsources of vegetable oils. Synthetic oils are also part of thisinvention and can include commercially available oils such as NEOBEE®and others. Squalene(2,6,10,15,19,23-Hexamethyl-2,6,10,14,18,22-tetracosahexaene) is anunsaturated oil which is found in large quantities in shark-liver oil,and in lower quantities in olive oil, wheat germ oil, rice bran oil, andyeast, and is a particularly preferred oil for use in this invention.Squalene is a metabolisable oil virtue of the fact that it is anintermediate in the biosynthesis of cholesterol (Merck index, 10thEdition, entry no.8619).

[0012] The oil in water emulsions which form part of the presentinvention when formulated with 3 D-MPL and QS21 are preferentialstimulators of IgG2a production and TH1 cell response. This isadvantageous, because of the known implication of TH₁ response in cellmediated response. Indeed in mice induction of IgG2a is correlated withsuch an immune response.

[0013] The observation that it is possible to induce strong cytolytic Tlymphocyte responses is significant as these responses, in certainanimal models have been shown to induce protection against disease.

[0014] The present inventors have shown that the combination of theadjuvants QS21 and 3D-MPL together with an oil in water emulsion with anantigen results in a powerful induction of CS protein specific CTL inthe spleen. QS21 also enhances induction of CTL on its own, while 3D-MPLdoes not.

[0015] Induction of CTh is easily seen when the target antigen issynthesised intracellularly (e.g. in infections by viruses,intracellular bacteria, or in tumours), because peptides generated byproteolytic breakdown of the antigen can enter the appropriateprocessing pathway, leading to presentation in association with class Imolecules on the cell membrane. However, in general, pre-formed solubleantigen does not reach this processing and presentation pathway, anddoes not elicit class I restricted CTL. Therefore conventionalnon-living vaccines, while eliciting antibody and T helper responses, donot generally induce CTL mediated Immunity. The combination of the twoadjuvants QS21 and 3D-MPL together with an oil in water emulsion canovercome this serious limitation of vaccines based or recombinantproteins, and induce a wider spectrum of immune responses.

[0016] CTL specific for CS protein have been shown to protect frommalaria in mouse model systems (Romero et al. Nature 341:323 (1989)). Inhuman trials where volunteers were immunised using irradiatedsporozoites of P. falciparum, and shown to be protected againstsubsequent malaria challenge, induction of CTL specific for CS epitopeswas demonstrated (Malik et al. Proc. Natl. Acad. Sci. USA 88:3300(1991)).

[0017] The ability to induce CTL specific for an antigen administered asa recombinant molecules is relevant to malaria vaccine development,since the use of irradiated sporozoites would be impractical, on thegrounds of production and the nature of the immune response.

[0018] In certain systems, the combination of 3D-MPL and QS21 togetherwith an oil in water emulsion have been able to synergistically enhanceinterferon γ production.

[0019] IFN-γ secretion is associated with protective responses againstintracellular pathogens, including parasites, bacteria and viruses.Activation of macrophages by IFN-γ enhances intracellular killing ofmicrobes and increases expression of Fc receptors. Direct cytotoxicitymay also occur, especially in synergism with lymphotoxin (anotherproduct of TH1 cells). IFN-γ is also both an inducer and a product of NKcells, which are major innate effectors of protection. TH1 typeresponses, either through IFN-γ or other mechanisms, providepreferential help for IgG2a immunoglobulin isotypes.

[0020] Particularly preferred adjuvants which may be used in theinvention described herein are combinations of 3D-MPL and QS21 (EP 0 671948 B1), oil in water emulsions comprising 3D-MPL and QS21 (WO 95/17210,PCT/EP98/05714), 3D-MPL formulated with other carriers (EP 0 689 454B1), or QS21 formulated in cholesterol containing liposomes (WO96/33739), or immunostimulatory oligonucleotides (WO 96/02555).

[0021] RTS is a hybrid protein comprising substantially all theC-terminal portion of the circumsporozoite (CS) protein of P.falciparumlinked via four amino acids of the preS₂ portion of Hepatitis B surfaceantigen to the surface (S) antigen of hepatitis B virus (HBV). Thestructure of RTS and the molecules from which it is derived is disclosedin International Patent Application Publication Number WO 93/10152.

[0022] When expressed in yeast RTS is produced as a lipoproteinparticle, and when it is co-expressed with the S antigen from HBV itproduces a mixed particle known as RTS,S.

[0023] Liver Stage Antigens are described in Malaria, Parasite Biology,Pathogenesis and Protection (1998 ASM Press, Washington D.C., edited byIrwin W. Sherman), especially Chapter 34 (P. Druilhe et al.).

[0024] A 26-amino acid synthetic peptide based on Plasmodium falciparumliver stage antigen 3 (LSA-3) is described in Eur J. Immunol., 1997, 27,1242-1253 (L. BenMohamed et al).

[0025] The immunogenicity of 12 synthetic peptides derived from four newPlasmodium falciparum molecules expressed at pre-erythrocytic stages ofthe human malaria parasite was reported in Vaccine 18 (2000), pages2843-2855 (L BenMohamed et al).

[0026] In these studies the adjuvant Montanide ISA-51 (SEPPIC, QuaiD'Orsay, France) was used. There is no report, however, of such peptidesbeing combined with other adjuvants. The present invention is based onthe surprising discovery that a Th-1 inducing adjuvant especially an oilin water emulsion which preferably comprises tocopherol, as such or incombination with QS21 and/or 3-D-MPL (or related molecules), enhancesimmune responses to a defined malaria antigen. Such enhancementavailable affords better immunological responses than hitherto before.

[0027] According to the present invention there is provided a vaccinecomposition comprising a Th1-inducing adjuvant in combination with aprotecting Liver Stage Antigen or immunological fragment thereof of ahuman malaria parasite with the proviso that when the immunologicalfragment is an immunological fragment of LSA-3, the Thl-inducingadjuvant is not Montanide.

[0028] In a preferred aspect of the invention the Th1-inducing adjuvantcomprises QS21, De-O-acylated monophosphoryl lipid A (3D-MPL) and an oilin water emulsion wherein the oil in water emulsion has the followingcomposition: a metabolisible oil, such a squalene, alpha tocopherol andtween 80.

[0029] Normally the vaccine composition according to any aspect of theinvention invokes a T cell response in a mammal to the antigen orantigenic composition and is preferably capable of stimulatinginterferon γ production. The oil in water emulsion used in the presentinvention may be utilised on its own or with other adjuvants orimmuno-stimulants and therefore an important embodiment of the inventionis an oil in water formulation comprising squalene or anothermetabolisable oil, alpha tocopherol, and tween 80. The oil in wateremulsion may also contain span 85 and/or Lecithin.

[0030] The combination of the two adjuvants QS21 and 3D-MPL togetherwith an oil in water emulsion is particularly preferred. This is knownand referred to herein as SBAS2, or alternatively simply as AS2 or AS02.

[0031] The ratio of QS21: 3D-MPL will typically be in the order of 1:10to 10:1; preferably 1:5 to 5:1 and often substantially 1:1. Thepreferred range for optimal synergy is 2.5:1 to 1:13D MPL: QS21.Typically for human administration QS21 and 3D MPL will be present in avaccine in the range 1 μg-100 μg, preferably 10 μg-50 μg per dose.Typically the oil in water will comprise from 2 to 10% squalene, from 2to 10% alpha tocopherol and from 0.3 to 3% tween 80. Preferably theratio of squalene: alpha tocopherol is equal or less than 1 as thisprovides a more stable emulsion. Span 85 may also be present at a levelof 1%. In some cases it may be advantageous that the vaccines of thepresent invention will further contain a stabiliser.

[0032] In an alternative preferred embodiment, the vaccine of theinvention may advantageously comprise a vesicular adjuvant formulationcomprising cholesterol, a saponin, and optionally an LPS derivative. Inthis regard the preferred adjuvant formulation comprises a unilamellarvesicle comprising cholesterol, having a lipid bilayer preferablycomprising dioleoyl phosphatidylcholine, wherein the saponin andoptionally the LPS derivative are associated with, or embedded within,the lipid bilayer. Preferably the vesicular adjuvant comprises both thesaponin and the LPS derivative. More preferably, these adjuvantformulations comprise QS21 as the saponin, and 3D-MPL as the LPSderivative, wherein the ratio of QS21:cholesterol is from 1:1 to 1:100weight/weight, and most preferably 1:5 weight/weight. Such adjuvantformulations are described in WO 96/33739 and EP 0 822 831 B, thedisclosures of which are incorporated herein by reference. For example asuitable formulation may contain 0.25 mg cholesterol, 1 mg dioleoylphosphotidylcholine, 5 ug 3D-MPL, and 50 ug QS21 and consist of smallamellar vesicles wherein the saponin (QS21) and the LPS-derivative(3D-MPL) are in the membranes of the vesicles.

[0033] It will be appreciated that variants or derivatives of QS21 and3-DMPL as described above may also be used without departing from thespirit of the invention.

[0034] The bacterial lipopolysaccharide derived adjuvants to beformulated in the adjuvant combinations of the present invention may bepurified and processed from bacterial sources, or alternatively they maybe synthetic. Accordingly, the LPS derivatives that may be used in thepresent invention are those inmmunostimulants that are similar instructure to that of LPS or MPL or 3D-MPL. In another aspect of thepresent invention the LPS derivatives may be an acylated monosaccharide,which is a sub-portion of MPL. In a preferred aspect the 3-DMPL is smallparticle 3-DMPL as described in WO 92/116556.

[0035] The oil emulsion adjuvants for use in the present invention maybe natural or synthetic, and may be mineral or organic. Examples ofmineral and organic oils will be readily apparent to the man skilled inthe art based on the description hereinabove.

[0036] Particularly preferred oil emulsions are oil in water emulsions,and in particular squalene in water emulsions.

[0037] In addition, the most preferred oil emulsion adjuvants of thepresent invention comprise an antioxidant, which is preferably the oilo-tocopherol (vitamin E, EP 0 382 271 B1).

[0038] WO 95/17210 discloses emulsion adjuvants based on squalene,x-tocopherol, and TWEEN 80, optionally formulated with theimmunostimulants QS21 and/or 3D-MPL.

[0039] The size of the oil droplets found within the stable oil in wateremulsion are preferably less than 1 micron, may be in the range ofsubstantially 30-600 nm, preferably substantially around 30-500 nm indiameter, and most preferably substantially 150-500 nm in diameter, andin particular about 150 nm in diameter as measured by photon correlationspectroscopy. In this regard, 80% of the oil droplets by number shouldbe within the preferred ranges, more preferably more than 90% and mostpreferably more than 95% of the oil droplets by number are within thedefined size ranges. The amounts of the components present in the oilemulsions of the present invention are conventionally in the range offrom 2 to 10% oil such as squalene; and when present, from 2 to 10%alpha tocopherol; and from 0.3 to 3% surfactant, such as polyoxyethylenesorbitan monooleate. Preferably the ratio of oil: alpha tocopherol isequal or less than 1 as this provides a more stable emulsion. Span 85may also be present at a level of about 1%. In some cases it may beadvantageous that the vaccines of the present invention will furthercontain a stabiliser. Preferably the oil emulsion contains a surfactantsuch as polyoxyethylene sorbitan monooleate (TWEEN80™), but it will beclear to the man skilled in the art that other surfactants may be used,preferred examples of which-are the SPAN series (especially SPAN85) andor lecithin.

[0040] The method of producing oil in water emulsions is well known tothe man skilled in the art. Commonly, the method comprises the mixingthe oil phase with a surfactant such as a PBS/TWEEN80™ solution,followed by homogerisation using a homogenizer, it would be clear to aman skilled in the art that a method comprising passing the mixturetwice through a syringe needle would be suitable for homogenising smallvolumes of liquid. Equally, the emulsification process in microfluidiser(M110S microfluidics machine, maximum of 50 passes, for a period of 2minutes at maximum pressure imput of 6 bar (output pressure of about 850bar)) could be adapted by the man skilled in the art to produce smalleror larger volumes of emulsion. This adaptation could be achieved byroutine experimentation comprising the measurement of the resultantemulsion until a preparation was achieved with oil droplets of therequired diameter.

[0041] In a preferred aspect of the invention the human malaria parasiteis Plasmodium falciparum.

[0042] In a particular aspect of the invention the said protecting LiverStage Antigen is the Liver Stage Antigen 3 (LSA-3) or immunologicalfragment thereof.

[0043] However other Liver Stage Antigens may also be used, for exampleLSA-1 and LSA-2 as described in Malaria, Parasite Biology, Pathogenesisand Protection (1998 ASM Press, Washington D.C., edited by Irwin W.Sherman), especially Chapter 34 (P. Druilhe et al.).

[0044] By immunological fragment is meant herein a molecule which has arelated or similar sequence to the reference antigen in terms of %homology and which can induce a similar immune response, cellular orhumoral, in vivo.

[0045] The LSA-3 antigen and polypeptide molecules containing at least10 consecutive amino acids of the amino acid sequence representing LSA-3are described in WO 96/41877. LSA-3 for use in the present invention maysuitably be prepared as described in the examples section of the presentspecification. Reference may also be made to C Marchand and P Druilhe,Bulletin of the World Health Organisation, Volume 68 (Suppl.) 158-164(1990) and U.S. Pat. No. 6,100,067.

[0046] In a further aspect there is provided a vaccine compositionaccording to the invention comprising in addition at least one otherprotecting antigen or an immunological fragment thereof, of a malariaparasite, in particular LSA-3.

[0047] In particular, the other malaria antigen may be selected from thefollowing group:

[0048] a) a hybrid protein comprising substantially all the C-terminalportion of the CS protein, four or-more tandem repeats of theimmunodominant region, and the surface antigen from hepatitis B virus(HBsAg), in particular RTS,S, or an immunogenic derivative includingfragments thereof;

[0049] b) the TRAP protein of the T9/96 isolate of Plasmodium falciparumand proteins having at least 80% homology thereto and immunogenicderivatives including fragments thereof (see European Patent ApplicationNo 91903249.0);

[0050] c) the MSP-1 of Plasmodium falciparum or Plasmodium vivax andproteins having at least 80% homology thereto and immunogenicderivatives including fragments thereof; and

[0051] d) the MSP-3 of Plasmodium falciparum or Plasmodium vivax andproteins having at least 70% homology with the C-terminal regionthereof, and immunogenic derivatives including fragments thereof.

[0052] MSP-1 of P.falciparum or P.vivax is described in U.S. Pat. No.4,837,016. Immunogenic derivatives include fragments thereof such as theC-terminal 42 KDa antigen (p42).

[0053] The MSP-3 antigen is described in U.S. Pat. No. 6,017,538.

[0054] Homology in sequence analysis may be established by the use ofBlast 2.0 and Fasta default settings of the algorithms used by theseprograms. The comparison of LSA-3 sequences in various isolates orstocks can be done using a calculation manual.

[0055] By C-terminal region of MSP-3 is meant a 185 amino acid regionfrom positions 193 to 381. It contains a leucine zipper on its extremity(C-terminus part) and is rich in acidic amino acids. Thethree-dimensional structure is coil-coiled. The clone DG 210 (aminoacids 193-257) corresponds to a globular region of high complexity andis followed by the coil-coiled region.

[0056] In a further aspect of the present invention there is provided avaccine as herein described for use in medicine.

[0057] In yet a further aspect the invention provides a process formaking a vaccine composition according to any aspect of the presentinvention by mixing the required components using standard techniques.Vaccine preparation is generally described in New Trends andDevelopments in Vaccines, edited by Voller et al., University ParkPress, Baltimore, Md., U.S.A. 1978.

[0058] In one aspect the process comprises admixing QS21, 3D-MPL and theoil in water emulsion with a protecting Liver Stage Antigen of a humanmalaria parasite as hereinabove defined, optionally with an additionalmalaria antigen.

[0059] The amount of protein in each vaccine dose is selected as anamount which induces an inmmunoprotective response without significant,adverse side effects in typical vaccinees. Such amount will varydepending upon which specific immunogen is employed and how it ispresented. Generally, it is expected that each dose will comprise 1-1000ug of protein, preferably 2-100 ug, most preferably 4-40 ug. An optimalamount for a particular vaccine can be ascertained by standard studiesinvolving observation of appropriate immune responses in subjects.Following an initial vaccination, subjects may receive one or severalbooster immunisation adequately spaced.

[0060] The formulations of the present invention maybe used for bothprophylactic and therapeutic purposes.

[0061] Accordingly in one aspect, the invention provides a method oftreatment comprising administering an effective amount of a vaccine ofthe present invention to a patient.

[0062] The following examples illustrate the invention.

EXAMPLES Example 1

[0063] Two adjuvant formulations were made each comprising the followingoil in water emulsion component.

[0064] SB26: 5% squalene 5% tocopherol 0.4% tween 80; the particle sizewas 500 nm size

[0065] SB62: 5% Squalene 5% tocopherol 2.0% tween 80; the particle sizewas 180 nm

[0066] 1(a) Preparation of Emulsion SB62 (2 Fold Concentrate)

[0067] Tween 80 is dissolved in phosphate buffered saline (PBS) to givea 2% solution in the PBS. To provide 100 ml two fold concentrateemulsion 5 g of DL alpha tocopherol and 5111 of squalene are vortexed tomix thoroughly. 90 ml of PBS/Tween solution is added and mixedthoroughly. The resulting emulsion is then passed through a syringe andfinally microfluidised by using an M110S microfluidics machine. Theresulting oil droplets have a size of approximately 180 nm.

[0068] 1(b) Preparation of Emulsion SB26

[0069] This emulsion was prepared in an analogous manner utilising 0.4,%tween 80.

[0070] To the emulsion of 1 a) or b) an appropriate amount of LSA-3 (forexample 2 μg to 100 μg) may be added and mixed. This may be combinedwith, for example, 50 μg/ml of 3D-MPL and 20 μg/ml of QS21 (or relatedmolecules) to give the final formulation.

Example 2

[0071] Protection Against Plasmodiums Falciparum Malaria in Chimpanzeesby Immunisation with a Conserved Pre-Erythrocytic Antigen, LSA-3

[0072] The basis of the strong immunological protection induced inhumans by vaccination with radiation-attenuated pre-erythrocytic malariaparasites is poorly understood. However it is now suspected that thetransformation of the irradiated sporozoites into live butdevelopmentally arrested intra-hepatic liver trophozoites is required toinduce protection⁹. This occurs at low (15-20 krad) but not at high(23-30 krad) irradiation doses^(9,10). We reasoned that the differentialresponse of hosts immunised with such irradiated sporozoites couldprovide a screen for molecules relevant to protection. We proceeded toscreen 120 phage lambda clones previously identified as expressing P.falciparum polypeptides that are expressed during pre-erythrocytic stageparasite development and which derive from ca. 20 distinctgenes^(6,7,11,12). A clone corresponding to each of these putative geneswas screened using eight sera from human volunteers ({fraction (4/6)}protected) and from chimpanzees (½ protected) immunised with sporozoitesirradiated at low or high doses. A single clone (DG729) reacted onlywith sera from protected humans and chimpanzees. This differentialreactivity was further confirmed with a peptide derived from thisfragment (Table I). This led us to select this clone for furtherinvestigation.

[0073] DG129 was used to probe a P. falciparum (K1) genornic library.One clone was found to contain the whole gene corresponding to DG729,and which was named Liver Stage Antigen-3 (LSA-3). Full description ofthe sequence, expression, location and conservation of the lsa-3 gene isprovided in the Supplementary Information (S.I.) and is summarised belowand in FIGS. 1-3. Briefly we identified a single-copy gene whichcomprises a mini-exon 1, a mini-intron, and a large exon 2 (FIG. 1a), astructure similar to that of other surface antigens of P. falciparum ¹³.It was recently confirmed that Lca-3 is located on chromosome 2¹⁴, wherethe gene was annotated as <<RESA-H3>> gene (Ace. Number AE001424).LSA-3, with a predicted molecular weight of 200 kDa (in K1), is made upof large non-repeated sequences flanking three glutamic acid-richrepeated regions, a feature that extends the known P. falciparumGlu-rich antigen network to include a pre-erythrocytic component Thelocation of the original fragment (DG729) and of the peptidescorresponding to the repeat region R2 and to the non-repetitive regionsNR-A and NR-B are shown in FIG. 1b. Naturally- or artificially-inducedantibodies against the non-repeated peptides and the recombinant proteinGST-PC were not cross-reactive with the repeated Glu-rich regions, andwere used for further studies.

[0074] Pre-erythrocytic expression of LSA-3 (see FIGS. 2-3 and see S.I.)was confutned a) by RT-PCR (primers i1 and i2) of total RNA and Westernblotting of protein extracts, isolated in both cases from sporozoites,and b) by inimunofluorescence antibody test (IFAT) on infected liversections and dry or wet sporozoite preparations, using antibodies to anon-crossreactive portion of the protein. In the five and six day-oldliver schizonts, LSA-3 was located in the parasitophorous vacuole and atthe periphery of maturing hepatic merozoites. This location isconsistent with the molecular structure of this protein, which containstwo hydrophobic regions (FIG. 1a). In our hands, mRNA from lsa-3 couldnot be detected in Northern blotted RNA from erythrocytic stages.Western blottings and IFAT of infected red blood cells were alsoconsistently negative with non cross-reactive antibodies. Reactivity washowever obtained when antibodies to the Glu-rich repeat region wereused. This might explain in part the detection of a putativelyhomologous antigen (D260) previously described in intra-erythrocyticparasites, and which was identified solely using antibodies whichcross-react extensively with Glu-rich epitopes¹⁶.

[0075] Polymorphism of many malaria vaccine candidate molecules is ofrecognised concern, we therefore investigated naturally occurringsequence variation in LSA-3 (see S.I.). The gene was consistentlydetected by PCR amplification of the NR-A region (primers S1 and S2) ina total of 111 P. falciparum isolates, strains or clones of variousgeographical origin. Using LSA-3 specific antibodies in IFAT assays, theexpression of LSA-3 was also detected in liver schizonts of two distinctstrains and in all the sporozoites from 30 wild isolates which developedin mosquitoes fed in vitro on Thai gametocytes. The repeat regions R1and R3 are highly conserved, but variation in the number and order ofthe repeal units of R2 was found to occur amongst different parasitelines. This did not however affect the predicted conserved ?-helicalorganisation, a secondary structure considered to be important indefining major B-cell epitopes since antibodies which recognise R2 didindeed react positively by IFAT with all the parasites tested. Thenon-repeated portions of exon 2, where numerous Th and CTL epitopes arefound¹⁷⁻¹⁹, displayed a remarkable degree of amino acid (an) sequenceconservation between different parasites (>95.5% homology). The sequenceof NR2 peptide was fully conserved amongst K1 and T9/96 parasites, thesource of the immunising proteins, the NF54 parasites used forsporozoite challenges, and 27 P. falciparum samples of variousgeographical origin¹⁷. An HLA-B53 restricted epitope identified in theNR-B region of LSA-3 (present in GST-PC recombinant protein) was alsofound to be free of variation in clone 3D7 and in 18 —Gambianisolates¹⁹. This conservation of inmiunologically important epitopescontrasts with substantial polymorphism in current pre-erythrocyticvaccine candidates.

[0076] We selected the chimpanzee to investigate the protective capacityof LSA-3 immunisation for the following reasons. The chimpanzee is theonly non-human primate fully susceptible to complete intra-hepaticdevelopment of P. falciparum, with a comparable rate of sporozoitetransformation to liver forms to that seen in humans⁹. The chimpanzee isalso the most closely related animal to humans (98.4% homology at theDNA level⁸), and one in which detailed investigations of immuneresponses can be performed and legitimately compared with those ofhumans¹⁷⁻¹⁸. The fact that parasitological and immunological events canbe directly examined in the liver biopsies, a possibility excluded forinfected humans, is clearly of considerable significance. A number ofpreliminary stringent tests were conducted in control animals in orderto validate the suitability of this model for vaccine evaluation. Sincecost and ethical considerations preclude the use of large number ofanimals, high reproducibility of the infection in this model system iscritical. In a preliminary experiment (Group I, Table II), we confirmedthat in the chimpanzee protection by inimnunisation with irradiatedsporozoite is radiation dose-dependent, and we validated the detectionof the infected red blood cells as an assay of protection. The resultsallowed us to define a number of important parameters: a) as in humans,chimpanzees develop a powerful protective response followingimmunisation with irradiated sporozoite, b) chimpanzees, like humans,remain broadly susceptible to at least five successive challenges, incontrast to lower primates or rodents which become refractory after thefirst challenge²⁰, and c) as a result of the high dose of inoculatedsporozoites detection of erythrocytic parasites Corresponded to thefirst invasion of red cells by merozoites released fromintra-hepatocytic schizonts. Positive blood smears were reproduciblyobtained in non-protected chimpanzees on days six or seven, In thechimpanzee erythrocytic infections normally remain sub-clinical andself-limiting which was in fact observed despite the high dosechallenges. These results have been recently confirmed in two furtherchimpanzees (Langermans J. et al, manuscript in preparation).

[0077] Having established the suitability of the chimpanzee, weproceeded to assay the protective value of LSA-3 immunisation bychallenge with viable P. falciparum sporozoites. In preliminaryexperiments, two animals were inimunised with a mixture of LSA-3 andLSA-1 recombinant proteins. Full protection against three challengesover several months was only seen in the animal which responded to LSA-3(both responded to LSA-1). In liver biopsies performed on this animal onday five, only one liver schizont of unhealthy appearance andinfiltrated by leukocytes could be detected in the 300 liver sectionsscreened (Dirk, FIG. 3). By contrast 2500 and 750 hepatic is schizontsof healthy appearance were observed in the two non-protected controls.

[0078] These results led us to focus further immunisation and challengeexperiments on LSA-3 alone. Two groups of chimpanzees were used toevaluate lipopeptide and recombinant protein formulations (Table II,Groups II-III). In Group II, one animal (Gerda) was initially imxnunisedsolely with the NR2 lipopeptide of LSA-3, and boosted by recombinantLSA-3 molecules in Montanide ISA 51. Gerda was fully protected whenchallenged with 10⁷ sporozoites, whereas the control receiving MontanideISA 51 was not (FIG. 4a).

[0079] In Gerda boosting with the recombinant LSA-3 formulation was notfound to induce any detectable increase in the strong B-cell, T-helpercell and CTL responses already evoked by the initial lipopeptide/peptideinjections^(17,18). We were therefore interested to see whether thesimple and well-tolerated peptidic formulation alone could induceprotection. Two chimpanzees, Mopia and Mgbado were imniunised with LSA-3lipopeptides/peptides alone (Table II, Group III). Protection against afirst challenge with 2×10⁴ sporozoites was obtained in both. The samegroup included an investigation of the effects of microbead presentationof recombinant proteins without adjuvant in one animal (Judy) whichresulted in a one-day delay to patency (FIG. 41,). Following asubsequent high dose sporozoite challenge (5×10⁶ sporozoites), bothMopia and Mgbado demonstrated a clear two-day delay to patency and a lowtransient parasitaemia, whilst no protection was found for Judy (FIG.4c). The delay to patency suggests that the immune responses had causeda reduction exceeding 90% of intra-hepatocytic schizont load²¹ (FIG. 4).

[0080] In chimpanzees from groups IV and V, we investigated the efficacyof a less complex lipopeptide mixture alone, or of recombinantsadjuvated by SBAS2, a novel adjuvant whose efficacy has been recentlyestablished in humans^(4,5). Since immunogenicity studies^(17,18) andanalysis of previous chimpanzee data had indicated that peptide CII waspoorly immunogenic and thus might not be critical, chimpanzee Patty wasimmunised by a mix of three instead of four peptides. This animal showedprotection upon challenge. Among four animals receiving SBAS2 adjuvatedLSA-3 proteins, two showed full, sterile protection against a mediumdose challenge. One showed a delay in patency which may be indicative ofpartial protection, whereas neither the fourth nor the control receivingSBAS2 adjuvant alone were protected. One of the two fully protectedchimpanzees was further challenged with a high dose three months laterand still showed full protection.

[0081] We present here the first description of protective vaccinationagainst human malaria in the chimpanzee. This model provided us withconvincing evidence that LSA-3 of P. falciparurn is a valuable candidatefor effective vaccination against pre-erythrocytic stages. A total ofnine animals were immunised using lipopeptides in saline or polypeptidesin either Montanide or SBAS2 adjuvants. Full sterile protection wasinduced in six of these nine chimpanzees on first challenge. If thesignificant delay as compared to controls is taken in consideration, aprotective effect induced by LSA-3 was shown in eight of nine animals.Out of the 14 challenges which were performed, complete protection wasobtained in seven, and partial protection in an additional fourchallenges. All seven control animals employed in these studies showeda-consistent pattern in the appearance and the course of the blood-stageparasitaemiae following each of the 12 challenges with viable parasites.Demonstration of this reproducibility in controls, in animals immunisedby over-irradiated sporozoites, and in an additional 26 challengesperformed in other experiments (not shown), is an essential point in theinterpretation of our data.

[0082] It is encouraging that protection was induced against aheterologous challenge (NF54) in outbred animals immunised with LSA-3molecules whose sequences were derived from K1 and T9/96 parasites. Avariety of imnmunisation strategies were investigated in the course ofthis work. The data underpin the value of the SBAS2 adjuvant. Theresults with Gerda, Mopia, Mgbado and Patty are also particularlyencouraging since they are based on simple peptide and lipopeptideformulations which are relatively easy to produce under GMPconditions²². In our animals no local or general reactions was detectedfollowing lipopeptide injections, an observation consistent withprevious experience with similar formulations derived from SW inmacaques²³ and HbS²⁴ or HIV²² in humans. This bodes well for futureclinical trials.

[0083] Methods

[0084] Selection of clone DG729. Dot blot analysis of theβ-galactosidase-fused-recornbinant proteins encoded by thepre-erythrocytic clones was performed on nitrocellulose as previouslydescribed⁷, using {fraction (1/100)} diluted human and chimpanzee scm.ELISA was performed in duplicate as previously described²⁵ on 1,100diluted sera using coating solutions of 0.3, 3 and 10 μg/ml of NR1, NR2and RE peptides respectively, in PBS.

[0085] LSA-3 cloning and characterisation. Detailed description ofmolecular methods, gene cloning, sequence data, protein characteristicsand description of the recombinant proteins and of the peptides areprovided in the S.I. The primers used for PCR: S1 (nucl.161-184)/S2(nucl.454-432) and for RT-PCR: i1 (nucl.695-722)/i2 (nucl.824-799),numbering refers to the lsa-3 sequence of K1 (Accession Nber AJ007010).All mouse sera used for the Western blot (at dilution {fraction(1/100)}) presented in FIG. 2 were obtained following 3 subcutaneousinjections of the ininiunogen (100 μg) emulsified in SBAS2 adjuvant⁴.Long synthetic peptides GP5, GP6, GP8 and GP11 were synthesised asdescribed in ref. 26 (see FIG. 1 for position).

[0086] Immunogens injected in chimpanzees. Sequences of the variousimmunogeris evaluated here consisted of clone DG729 and inserts NN andPC, as well as peptides (pep.) NR1, NR2, RE and CT1; their location isshown in FIG. 1 and described in more details in the S.I. Clone DG729,as well as inserts NN and PC were expressed asglutathione-S-transferase-fused recombinants and purified according tomanufacturer recommendations (Invitrogen, The Netherlands). RecombinantsGST-DG729,-NN and -PC were designed so as to cover 95% of the LSA-3antigen and were used as a mixture mentioned as LSA-3 GST-rec. PeptidesNR1, NR2 and CT1, were also synthesised as pahnitoyl-conjugatedlipopeptides (lipopep.), as described in ref. 17. Combination ofsynthetic compounds (mentioned as (lipo)pep.) consisted in a mixture ofNR1, NR2 and CT1 lipopeptides and of RE peptide. All peptides andlipopeptides were purified to >90% purity by reversed-phasechromatography, and the impurities consisted essentially of relatedpeptides of shorter sequences¹⁷.

[0087] Chimpanzee immunisations and challenges. None of the chimpanzeesincluded in this study hail previously been exposed to malariainfections or malarial antigens. Recombinant and synthetic compoundswere-injected subcutaneously, at a dose of 100 μg for each peptideand/or lipopeptides, and/or 50 μg for each protein. Lipopeptides werealways injected in PBS and, except when mentioned, peptides andrecombinants were emulsified in Montanide ISA51. Group I animals (Carland Japie) were immunised by five intra-venous injections of 5×10⁶gamma-irradiated sporozoites at day 0 and weeks 8, 24, 44 and 65, andreceived three challenges at weeks 71, 97 and 123 (challenge doses aregiven in Table II). One year after the three challenges reported here,these chimpanzees were re-immunised once, and received one low and onehigh dose challenges, which revealed the same pattern of protection (notshown, Langermans J. et al., manuscript in preparation). In Group II,Gerda received NR2 lipopeptide at day 0 and weeks 3, 13 and 31 asdescribed in ref. 17. She was then boosted with the mixture of LSA-3GST-rec. at weeks 40,45,48 and 50. Control animal Lianne receivedMontanide ISA51. Challenges were performed at week 60. Group III animalswere immunised at day 0 and weeks 3 and 6. Mopia and Mgbado receivedLSA-3 (lipo)peptides whereas Judy was injected with LSA-3 (3ST-rec.adsorbed to latex miorobeads. Challenges LD and HD were performed atweeks 21 and 29. In Group IV, Patty received LSA-3 (lipo)peptides, butwithout lipopeptide CT1, whereas Wendy and Willy were injected withLSA-3 GST-rec in SBAS2 adjuvant^(4,5). Control animal Helen receivedSBAS2 adjuvant only. All animals were immunized at weeks 0, 4 and 8 andwere challenged with 20,000 sporozoites at week 13. In Group V, Cindyand Marty were both immunised at weeks 0, 4, 8 and 26 with TSA-3 GST-recin SBAS2 adjuvant (as in Group IV) and negative control animal Fauzireceived over-irradiated sporozoites similarly to Sapie (Group I) atweeks 5, 8, 11 and 26. Challenges LD and HD were performed at weeks 33and 46 in all three animals.

[0088] NF54 sporozoites were obtained from dissected salivary glands ofinfected Anopheles gambiae as previously described²⁷. Sporozoites werepooled, resuspended in PBS and injected intravenously. All animals ineach group were challenged with the sante pool of sporozoites. For costreasons, extensive evaluation of the Minimal Infective Dose has not beenundertaken, however challenge with 5×10³ sporozoites, the lowest doseused to date, has proven infective in four other animals (Thomas, A. W.,unpublished data).

[0089] Determination of the protective status. For Groups I, II, IV andV, animals blood was taken on days five to nine, and evaluated by thickand thin film Gienisa-stained preparations, and confirmed in all casesby in vitro culture (not shown), as described in ref. 21. For Group IIIchimpanzees blood taken every day from day five up to day 18, then everyother day up to day 30, was used to prepare thin and thick smears whichwere Giemsa-stained and examined by two separate microscopists. Achimpanzee was considered a) totally protected when no parasites couldbe detected in the circulation blood, by direct microscopicalobservation and by long term culture, or b) partially protected whentime to patency was delayed by one or more days as compared to thatobserved in control animals. In mice, these delays correspond to aprotection of 80% (24 h) or 96% (48 h) against sporozoite challenges. Inhumans, a 12 hour delay was calculated to correspond to a 92% reductionof liver forms following sporozoite challenges²¹. In a limited number ofanimals a liver biopsy was performed under anaesthesia by a veterinarydoctor on day five following a high dose challenge. Material was fixedand 4 μm sections were made and stained by Gietnsa-collophonium²⁸ beforecomplete microscopic enumeration of the liver forms in 300 sections(average area 0.8 cm²). All animals were curatively treated withchloroquine immediately after the period of observation, andirrespective of their protective status.

REFERENCES TO EXAMPLE 2

[0090] 1. Herrington, D., el al. Successful immunization of humans withirradiated malaria sporozoites: humoral and cellular responses of theprotected vaccinees. Am. J. Trop. Med. Hyg. 45, 539-547 (1991).

[0091] 2. Egan, I. E., et al. Humoral immune responses in volunteersimmunized with irradiated Plasmodium falciparum sporozoites. Am. J.Trop. Med Hyg. 49, 166-73 (1993).

[0092] 3. Facer, C. A. & M., Tanner. Clinical trials of malariavaccines: progress and prospects. Adv. Parasitol. 39, 1-68 (1997).

[0093] 4. Stoute, J. A., et al. A preliminary evaluation of arecombinant circumsporozoite protein vaccine against Plasmodiumfalciparum malaria. New Engl. J. Med. 336, 86-91(1997).

[0094] 5. Stoute, J. A., et al. Long-term efficacy and immune responsesfollowing immunization with the RTS,S malaria vaccine. J. Infect Dis.178, 113944 (1998).

[0095] 6. Guérin-Marchand, C., et al. A liver stage-specific antigen ofPlasmodum falciparum characterized by gene cloning. Nature. 329, 164-167(1987).

[0096] 7. Marchand, C. & Druilhe, P. How to select Plasmodium falciparumpre-erythrocytic antigens in an expression library without definedprobe. Bull. WHO. 68 (suppl.), 158-164 (1990).

[0097] 8. Miyaxnoto, M. M., Koop, B. F., Slightom, I. L., Goodman, M.and M. R., Tennant. Molecular systematics of higher primates:genealogical relations and classification. Proc. Nat. Acad Sci. U.S.A.85, 7627-31 (1988).

[0098] 9. Druilhe, P., et al. in “Malaria. Parasite Biology,Pathogenesis and Protection” (eds. Irwin W. Sherman), p.513-543(American Society for Microbiology, Washington D.C., 1998).

[0099] 10. Mellouk, S., Lunel, F., Sedegah, M., Beaudoin, R. L. and P.,Druilhe. Protection against malaria induced by irradiated sporozoites.Lancet. 335, 721 (1990). the circuntsporozoite protein retardsinfection. J. Gun. Microbial. 27, 1434-1437 (1989).

[0100] 22. Gahery-Segard, H., et cd. Multiepitopic B- and I-cellresponses induced in humans by a Human Immunodeflciency Virus type 1lipopeptide vaccine. J. Viral. 4, 1694-703 (2000).

[0101] 23. Bourgault, I., et al. Simian immunodeficiency virus as amodel for vaccination against HIV: induction in rhesus macaques of GAGor NEF specific cytotoxic T lymphocytes by lipopeptides. J. Immunol.152, 2530-2537 (1994).

[0102] 24. Vitiello, A., et al. Development of a lipopeptide-basedtherapeutic vaccine to treat chronic HBV infection. Induction of aprimary cytotoxic T lymphocyte response in humans. J. Clin. Invest. 95,341-349 (1995).

[0103] 25. Londoño, J. A., Gras-Masse, H., Dubeaux, C., Tartar, A. andP., Druilhe. Secondary structure and immunogenicity of hybrid syntheticpeptides derived from two Plasmodium falciparum pre-erythrocyticantigens. J. Immunol. 145, 1557-1563 (1990).

[0104] 26. Roggero, M. A., et al. Synthesis and immunologicalcharacterization of 104-mer and 102-mer peptides corresponding to the N-and C-terminal regions of the Plasmodium falciparum CS Protein. Mol.Jmmunol. 32, 1301-1309 (1995).

[0105] 22. Ponnudurai, T., at al. Sporozoite load of mosquitoes infectedwith Plasmodium falciparum. Trans. Roy Soc. Trap. Med Hyg. 83, 67-70(1989).

[0106] 28. Druilhe, P., Puebla, R. M., Miltgen, P., Perrin, L. and M.,Gentilini. Species- and stage-specific antigens in exoerythrocyticstages of Plasmodium falciparum. Am. J. Trap. Med Hyg. 33, 336-341(1984).

[0107] 29. Meis, J. F. G. M., et al. Plasmodium falciparum: studies onmature exoerytbrocytic forms in the liver of the chimpanzee, Pantroglodytes. Exp. Parasitol. 79,1-11(1990).

Example 3

[0108] The following experiments take advantage of the long peptidestrategy (LSP) developed by GianPietro Conadin in Lausanne, which allowone to establish proof of

[0109] 11. Fidock, D. A., et al. Cloning and characterization of aPlasmodium falciparum sporozoite surface antigen—STARP. Mol. Biochem.Parasitol. 64, 219-232 (1994).

[0110] 12. Bottius, E. et al. A novel Plasmodium falciparum sporozoiteand liver stage antigen (SALSA) defines major B, I helper, and CTLepitopes. J. Immunol. 156, 2874-2884 (1996).

[0111] 13. Kemp, D. J., Cowman, A. F. and D., Walliker. Geneticdiversity in Plasmodium falciparum. Adv. Parasitol. 29, 75-149 (1990).

[0112] 14. Gardner, M. J., at al. Chromosome 2 sequence of the humanmalaria parasite Plasmodium falciparum. Science, 282, 1126-1132 (1998).

[0113] 15. Moelans, I. I. M. D. & J. G. G., Schoenmakers. Crossreactiveantigens between life cycle stages of Plasmodium falciparum. Parasitol.Today. 8, 118-123(1992).

[0114] 16. Barnes, D. A., Wollish, W., Nelson, R. G., Leech, J. H. andC., Petersen. Plasmodium falciparum: D260, an intraerythrocytic parasiteprotein, is a member of the glutamic acid dipeptide-repeat family ofproteins. Exp. Parasitol., 81, 79-89 (1995):

[0115] 17. Ben Mohamed, L., et al. Lipopeptide immunization withoutadjuvant induces potent and long-lasting B, I helper, and cytotoxic Tlymphocyte responses against a malaria liver stage antigen in mice andchimpanzees. Eur. J. Immunol. 27, 1242-1253 (1997).

[0116] 18. Ben Moharned, L. et al. High immunogenicity in chimpanzees ofpeptides and lipopeptides derived from four new Plasmodium falciparumpre-erythrocytic molecules. Vaccine, 18, 2843-2855 (2000).

[0117] 19. Aidoo, M., et al. CTL epitopes for HLA-B53 and other HLAtypes in the malaria vaccine candidate Liver Stage Antigen-3. Infect.Immun. 68, 227-232 (2000).

[0118] 20.Nüssler, A. K., et al. In viva induction of the nitric oxidepathway in hepatocytes after injection with irradiated malariasporozoites, malaria blood parasites or adjuvants. Eur. J. Immunol. 23,882-887 (1993).

[0119] 21. Murphy, J. R., Baqar, S., Davis, J. R., Herrington, D. A. andD. F., Clyde. Evidence for a 6.5-day minimum exoerythrocytic cycle forPlasmodium falciparum in humans and confirmation that immunization witha synthetic peptide representative of a region of concept at clinicallevel by producing in short time and at low cost Long SyntheticPeptides. These are in fact short proteins which can be employed inclinical trials. A series of 17 overlapping Long Synthetic Peptides wassynthesised covering the full length of the LSA-3 molecule.

[0120] These peptides were used in antigenicity studies at T-cell andB-cell level in exposed individuals in the field in Senegal to monitorantibody and lymphoproliferative responses to each of them in exposedpopulations. They were used also to immunise mice using AS2 adjuvant.

[0121] Both studies demonstrated a very strong antigenicity of most ofthe peptides which, each, defined at least one B-cell and one T-cellepitope and immunogenicity studies in mice indicated that most peptidesstudied were also strongly immunogenic to laboratory mice (summarisedin: Perlaza at al. European Journal of Immunology, 2001 Jul;31,7,2200-9)

[0122] Challenge experiments with the cross-reactive Plasmodium ofrodents, Plasmodium yoelii, indicated in particular that a peptidecalled GP1 could induce protection against virulent P.yoelii sporozoitechallenge. For further studies in humans, to chose the irnmunizinggpeptides we relied on initial results obtained with the recombinantdenominated DG729 which overlaps part of the non-repetitive N-terminalregion of the molecule and the beginning of the repeat region.

[0123] Two types of formulations were investigated:

[0124] a) A very long LSP of ca 160 aminoaeids, covering the end of theNon-repeated region, including the short peptides NR1 and NR2investigated formerly and the beginning of the repeat region.

[0125] b) A mix of 2 peptides, one covering only the non-repeat region,called GP1 and another, called GP14, located in the beginning of therepeat region. For practical reasons, it was found that it would bedifficult to produce in sufficiently pure form the very long speciesmentioned above in a), and that for GMP production it would be safer torely on a mix of the two peptides mentioned in b), namely GP1 and GP14.

[0126] Therefore, pre-clinical studies were performed in South-americanprimates, Aotus trivirgatus griseimembra, by Blanca-Liliana Perlaza inthe collaborative laboratory of Socrates Herrera in Cali, Colombia.

[0127] 7 Animals were Enrolled in this Study as Follows:

[0128] Group 1: 2 animals receiving 3 injections of LSA-3 GPI LSP at adose of 50 mg pet injection per animal, adjuvated by AS2 in a totalvolume of 500 ml per injection.

[0129] Group 2: 2 animals receiving 3 injections of a mixture ofpeptides LSA-3 GP1 SA-3 GP14, at a dose of 50 mg of each peptide perinjection per animal, adjuvated by AS2 in a volume of 500 ml perinjection.

[0130] Group 3: 2 animals receiving 3 injections of PBS with adjuvantAS2 in a volume of 500 ml per injection, plus ] non-immunised control.

[0131] One month after the-last immunisation, which were well toleratedand did not induce any major local or general reactions, blood sampleswere taken to analyse immunogenicity: results are shown in thecorresponding graphs and demonstrated strong antibody production,lymphoproliferative responses and Interferon-g production, both inculture supernatant of lymphocytes and by Elispot technique. Animalswere challenged by intra-venous inoculation of 100 000 sporozoites ofthe Santa Lucia strain of Plasmodium falciparium 3 months after the lastimmunisation Blood samples taken over a period of 60 days afterchallenge may be analysed by 3 different techniques, namely microscopyof coloured blood, Polymerase Chain Reaction and LDH assay.

[0132] The study of the degree of protection achieved by the LDH assayhas been completed. This method relies on the detection of the parasiteby a double-site ELISA capture assay which has been recently described(Druilhe et al., American Journal, 64 (5, 6) 2001, 233-241) and whichwas shown to be at least 10 times more sensitive than microscopy. Theresults obtained are presented in the figures. They essentially showthat the 3 control animals became blood-stage positive, i.e. yieldedpositive parasite-specific LDH detection during the follow-up, whereasthe 2 immunised groups remained consistently negative by this techniqueover the 60 days of follow-up.

[0133] The results support the protection induced by immunisation by theGP1 LSP or the GP1+GP14 LSPs adjuvanted by AS2. These results are inagreement with previous data obtained using the recombinant DG729 alonewhich covers the same region of the antigen, as well as immunisationperformed by a mix of lipopeptides coveting the same region, as well asthose obtained by a mix of 3 recombinants (729, NN, PC) adjuvanted byAS2 (Daubersjes et al., Nature Medicine, Nov. 2000, 6, 11, 1258-1263).The sequence of the 2 peptides employed is shown below. GP1 L A S E E VK E K I L D L L E E G N T L T E S V D D N K N L E E A E D I K E N I L LS N I E E P K E N I I D N L L N N I G Q N S E K Q E S V S E N V Q V S DE L F N E L L N S V D V N G E V K E N I L E E S Q V N D D I F N S L V KS V Q Q E Q Q H N GP 14 ESVAENVEES VAENVEEIVAPTVEEIVAPTVEEIVAPSVVESVAPSVEES VEENVEESVA ENVEESVAEN VEESVAENVEESVAENVEEI VAPTVE

[0134] Code Spz. IFAT NR2 or irrad. titers peptide Name dose on spz.status (aa 198-223) V4 23.6 4,096 not protected 0.5 V5 23.6 32,000 2 daydelay 0.5 Japie 30 3,200 not protected 0.7 V6 20.8 5,120 Protected 3.8V7 20.8 41,960 Protected 2.6 V8 20.8 40,960 Protected 4.8 WR4 15 3,200Protected 3.4 Carl 18 6,400 Protected 2.3

[0135] Table I (above): Differential reactivity of sera from protectedor non-protected humans or chimpanzees with peptide NR2. IgG-specificantibodies against peptide NR2 were measured by ELISA in sera from humanvolunteers (codes) and chimpanzees (names in italic) immunised withsporozoites irradiated at low or high dose (in krad). Codes,immunisation schemes, sporozoite IFAT titres and protective statusdetermination for human volunteers V4-V8 and WR4 are detailed in ref. 1and 2, respectively. Chimpanzees Carl and Japie were inirnunised andchallenged as described in the text and the Methods (Group I). ELJSAtitres are expressed in arbitrary units representing the ratio of themean ODs from test sera to the mean OD plus three standard deviationsfrom 10 controls studied in parallel iii the same plate. Results aretaken as positive for ratios above one (in bold). Similar experimentsperformed with peptides NR1 and RE (see FIG. 1) yielded negative resultswith these sera (not shown). PROTECTION ANIMAL GROUPS Immunisation andchallenge LD HD Chimp. Immunisation protocols^(a) dates (weeks) 2 × 10⁴10⁷ Carl Japie Marcel Theo GroupI^(b 18 krad-irradiated sporozoites 30 krad-irradiated sporozoites unimmunised control unimmunised control)

+−−− +−−− Gerda Lianne Group II [lipopep. NR2] then [GST-rec. inISA51]control ISA 51

nd nd +− Mopia Mgbado Judy Ondele Makata Group III[(lipo)pep.][(lipo)pep.][GST-rec./microbeads]control GST/microbeadsunimmunised control

++d1 −− d2 d2 −−− Group IV Patty [(lipo)pep.]^(d) + nd Wendy [GST-rec.in SBAS2] + nd Willy [GST-rec. in SBAS2] − nd Helen control SBAS2 − ndGroup V Cindy [GST-rec. in SBAS2] + + Marty [GST-rec. in SBAS2] d1 −Fauzi 30 krad-irradiated sporozoites − −

[0136] Table II (above): Immunisation and challenge experiments in thechimpanzees. Challenges were performed with either 2×10⁴ (low dose) or10⁷ (high dose) NF54 P. falciparium sporozoites (“Protection” column).Immunisation schedules (in brackets under the bar) and of challenges(indicated by arrows above the bar) are expressed in weeks from firstimmunisation. Complete protection is indicated with (α); a delay topatency (in days) as compared to controls and non-protected animals isindicated by d1 or d2 (determination of the protective status isdetailed in the Methods).

LEGENDS FOR FIGURES

[0137]FIG. 1: Schematic representation of the LSA-3 gene, recombinantproteins and peptides. a) 6.2 Kb Eco RI-insert isolated from K1 parasitegenomic DNA library that hybridised with DG729. The 5.53 Kb genecomprises a 198 bp exon 1, a 168 bp intron (i) and a 5.16 Kb exon 2.Regions NR-A, -B and -C correspond to non-repeated sequences whereas R1to R3 designate the three repeat blocks. The two hydrophobic regionspotentially corresponding to the NH2-terminal signal peptide and theanchor region are indicated by HR1 and HR2 respectively. b) Location ofthe sequences encoding for LSA-3 in the recombinant fusion proteins(first line) and the synthetic peptides (strokes) used in this study(see Supplementary Information for aa numbering). For the immunisations,CT1 and NR2 were also used as palmltoyl-conjugated lipopeptides¹⁷(indicated by *).

[0138]FIG. 2: LSA-3 expression in P. falciparium sporozoites. Westernblot analysis was performed on protein extracts from NF54 sporozoitesand control uninfected mosquito salivary glands using mouse antiseradirected against: C) control GST, I) GST-PC, 2) peptides GP5, GP6, GP8or GP11, 3) GST-729 (see FIG. 1, Methods and S.I.). LSA-3 is visualisedas a 175 kDa protein (*), in agreement with the theoretical molecularweight of LSA-3 in this parasite strain.

[0139]FIG. 3: Immunostaining of P. falciparium pre-erythrocytic stageswith anti-LSA-3 antibodies. a) sporozoites stained by IFAT with humanantibodies affinity purified on recombinant βgal-DG729. b) staining byIFAT of day six post-challenge liver stages from a chimpanzee, using theantibodies induced by lipopeptide NR2 injection¹⁷ in chimpanzee Gerda(see S.I. for additional pictures). c) The single residual liverschizont detected in a chimpanzee Dirk (day five post-challenge)appeared infiltrated by lymphomononuclear cells, as compared in d) toone of the numerous healthy schizonts observed in the control chimpanzeePeer (total of ca 2500 scbizonts/300 liver sections. Giemsa-collophoniumstaining²⁸) (see text). Bars correspond to 5 μm in panel a) and 20 μm inpanels b) to d).

[0140]FIG. 4: Blood parasitaemia courses in Groups II and III. a)chimpanzees from Group II and b-c) animals in Group III, following highdose (HID) or low dose (LD) challenges with NF54 sporozoites. Names oftotally or partially protected animals are in bold. Hatched patternscorrespond to control chimpanzees. Parasitaemia scales are different foreach challenge, as expected from challenges with different numbers ofsporozoites. Note that the day of patency in control and non-protectedanimals was the same for a given challenge inoculum within each group(in the above and in other groups not shown here).

[0141]FIG. 5: antibody titers following immunisation with GP1 and GP14

[0142] The figure shows the results from ELISA assays of Aotus M73 andD114 immunised by LSA-3 GP1+adjuvant AS2 against the immunising peptideGP1 or the recombinant DG729. In both cases, the titers are high as theresult is significant for values higher than an ELISA ratio of 1. Thesecond half of the figures show the results obtained in Aotus M88 andM91 inimunised by GP1+GP14 adjuvated by AS2, against peptide GP1 andGP14 or the recombinant 729 or NN covering the repeat region, or acontrol recombinant (GST). Here again, the responses are high againstboth immunising peptides.

[0143]FIG. 6: Proliferative Responses to GP1 and GP14

[0144] The figure shows the proliferative responses in M88 and M91 andM223 (a third animal, included in fact in group 2, but not challenged)of monkeys immunised by a mix of GP1+GP14 with AS2 adjuvant. Significantproliferative responses were obtained to the immunising peptide GP1 and,to a lesser extent, GP14, or to smaller peptides here contained in thelonger ones such as NR1 NR2, or the sporozoites themselves (Pf).However, responses were lower and borderline (threshold of positivity=2)in monkey M88 as compared with the 2 others. The PHA is a positivestimulation control.

[0145] In monkeys immunised only by GP1 adjuvanted by AS2, positiveresponses were mainly recorded in monkey M73 and were only borderlinepositive to the immunising peptide GP1 in monkey V114 (whereas they areessentially negative in monkey M51).

[0146]FIG. 7: Elispot Assays

[0147] The figure shows responses recorded in monkey M73 and V114receiving GP1 peptide and monkeys M88 and M91 receiving the mix ofGP1+GP14. The results are expressed as a mean of SFCs, i.e. of coloniessecreting Interferon-g in an Elispot assay. Results are stronglypositive in all monkeys towards several peptides, e.g. the recombinant729 and immunising peptides GP1 in M73, as well as P. falcipariumsporozoites, most peptides or sporozoites employed in monkey V114, therecombinmits 729 and NN for monkey M8S and M91 as well as the immunisingpeptide GP1 and, to a lesser extent GP14 in the same monkeys, as well asP. falciparium sporozoites in the same monkeys.

[0148]FIGS. 8a-d: LDII levels

[0149] The figure shows the various levels of LDH detected in thevarious monkeys mentioned above, as compared to the controls (blueline). The horizontal line is the threshold of positivity determined asthe mean OD value in controls+3 standard the assay determined as themean value given by uninfected aotus control blood+3 standard deviations(results below this threshold value are negative and results above thisthreshold value are positive). The horizontal axis indicates the daysfollowing-sporozoite injection, when samples were taken and processed inthe DELI-LDH assay.

EXAMPLE 4

[0150] Sequence Data and Supplementary Information

[0151] The following further information exemplifying the invention issupplied:

[0152] Sequence Data—Gene: full Sequence (K1 parasite)

[0153] Protein: full Sequence (K1 parasite)

[0154] Clones DG729/DG679 (T9/96 parasite)

[0155] Note on LSA-3 sequence in parasite 3D7

[0156] Gene & Protein—Structure. Restriction map. Hydrophobicity

[0157] Oligonucleotides employed

[0158] Organisation

[0159] Regions & Comments—NR-A.R1.R2.NR-B.R3.NR-C

[0160] Conservation—of the gene

[0161] of the sequence

[0162] of repeat region R2

[0163] comparaison of immunising and challenging sequences

[0164] Stage Specificity & Subcellular Location

[0165] Homologies—Intraspecies

[0166] Interspecies

[0167] Synthetic Peptides & Recombinant Proteins used for ChimpanzeeImmunisations

[0168] Peptides CT1.NR1.NR2.RE

[0169] Recombinant proteins B-729. GST-729. OST-NN. GST-PC

[0170] Methods

[0171] References to Example 4

[0172] Full sequence listings in the appropriate format are alsoprovided herein.

1 10 1 5528 DNA K1 Parasite Strain 1 atgacaaata gtaattacaa atcaaataataaaacatata atgaaaataa taatgaacaa 60 ataactacca tatttaatag aacaaatatgaatccgataa aaaaatgtca tatgagagaa 120 aaaataaata agtacttttt tttgatcaaaattttgacat gcaccatttt aatatgggct 180 gtacaatatg ataataacgt aagataaaaaactaaataat aaatataaat aaaaaaaaaa 240 aaaaaaaaaa aaaaatcaac tatatagtatgtataatata tatatatata tatatatata 300 tatatatata tatatattta tttttatttatttattaatt tttttttttt tatattatct 360 ttttagtctg atataaacaa gagttggaaaaaaaatacgt atgtagataa gaaattgaat 420 aaactattta acagaagttt aggagaatctcaagtaaatg gtgaattagc tagtgaagaa 480 gtaaaggaaa aaattcttga cttattagaagaaggaaata cattaactga aagtgtagat 540 gataataaaa atttagaaga agccgaagatataaaggaaa atatcttatt aagtaatata 600 gaagaaccaa aagaaaatat tattgacaatttattaaata atattggaca aaattcagaa 660 aaacaagaaa gtgtatcaga aaatgtacaagtcagtgatg aactttttaa tgaattatta 720 aatagtgtag atgttaatgg agaagtaaaagaaaatattt tggaggaaag tcaagttaat 780 gacgatattt ttaatagttt agtaaaaagtgttcaacaag aacaacaaca caatgttgaa 840 gaaaaagttg aagaaagtgt agaagaaaatgacgaagaaa gtgtagaaga aaatgtagaa 900 gaaaatgtag aagaaaatga cgacggaagtgtagcctcaa gtgttgaaga aagtatagct 960 tcaagtgttg atgaaagtat agattcaagtattgaagaaa atgtagctcc aactgttgaa 1020 gaaatcgtag ctccaagtgt tgtagaaagtgtggctccaa gtgttgaaga aagtgtagaa 1080 gaaaatgttg aagaaagtgt agctgaaaatgttgaagaaa gtgtagctga aaatgttgaa 1140 gaaagtgtag ctgaaaatgt tgaagaaagtgtagctgaaa atgttgaaga aatcgtagct 1200 ccaactgttg aagaaatcgt agctccaactgttgaagaaa ttgtagctcc aagtgttgta 1260 gaaagtgtgg ctccaagtgt tgaagaaagtgtagaagaaa atgttgaaga aagtgtagct 1320 gaaaatgttg aagaaagtgt agctgaaaatgttgaagaaa gtgtagctga aaatgttgaa 1380 gaaagtgtag ctgaaaatgt tgaagaaagttagctgaaaa tgttgaagaa atcgtagctc 1440 caactgttga agaaatcgta gctccaactgttgaagaaat tgtagctcca agtgttgtag 1500 aaagtgtggc tccaagtgtt gaagaaagtgtagaagaaaa tgttgaagaa agtgtagctg 1560 aaaatgttga agaaagtgta gctgaaaatgttgaagaaag tgtagctgaa aatgttgaag 1620 aaagtgtagc tgaaaatgtt gaagaaagtgtagctgaaaa tgttgaagaa agtgtagctg 1680 aaaatgttga agaaagtgta gctgaaaatgttgaagaaat cgtagctcca actgttgaag 1740 aaatcgtagc tccaactgtt gaagaaattgtagctccaag tgttgtagaa agtgtggctc 1800 caagtgttga agaaagtgta gaagaaaatgttgaagaaag tgtagctgaa aatgttgaag 1860 aaagtgtagc tgaaaatgtt gaagaaagtgtagctgaaaa tgttgaagaa agtgtagctg 1920 aaaatgttga agaaatcgta gctccaactgttgaagaaat cgtagctcca actgttgaag 1980 aaattgtagc tccaagtgtt gtagaaagtgtggctccaag tgttgaagaa agtgtagaag 2040 aaaatgttga agaaagtgta gctgaaaatgttgaagaaag tgtagctgaa aatgttgaag 2100 aaagtgtagc tgaaaatgtt gaagaaatcgtagctccaac tgttgaagaa atcgtagctc 2160 caactgttga agaaattgta gctccaagtgttgtagaaag tgtggctcca agtgttgaag 2220 aaagtgtaga agaaaatgtt gaagaaagtgtagctgaaaa tgttgaagaa agtgtagctg 2280 aaaatgttga agaaagtgta gctgaaaatgttgaagaaag tgtagctgaa aatgttgaag 2340 aaatcgtagc tccaactgtt gaagaaatcgtagctccaac tgttgaagaa attgtagctc 2400 caagtgttgt agaaagtgtg gctccaagtgttgaagaaag tgtagaagaa aatgttgaag 2460 aaagtgtagc tgaaaatgtt gaagaaagtgtagctgaaaa tgttgaagaa agtgtagctg 2520 aaaatgttga agaaagtgta gctccaactgttgaagaaat tgtagctcca agtgttgaag 2580 aaagtgtagc tccaagtgtt gaagaaagtgttgctgaaaa cgttgcaaca aatttatcag 2640 acaatctttt aagtaattta ttaggtggtatcgaaactga ggaaataaag gacagtatat 2700 taaatgagat agaagaagta aaagaaaatgtagtcaccac aatactagaa aacgtagaag 2760 aaactacagc tgaaagtgta actacttttagtaacatatt agaggagata caagaaaata 2820 ctattactaa tgatactata gaggaaaaattagaagaact ccacgaaaat gtattaagtg 2880 ccgctttaga aaatacccaa agtgaagaggaaaagaaaga agtaatagat gtaattgaag 2940 aagtaaaaga agaggtcgct accactttaatagaaactgt ggaacaggca gaagaaaaga 3000 gcgcaaatac aattacggaa atatttgaaaatttagaaga aaatgcagta gaaagtaatg 3060 aaaatgttgc agagaattta gagaaattaaacgaaactgt atttaatact gtattagata 3120 aagtagagga aacagtagaa attagcggagaaagtttaga aaacaatgaa atggataaag 3180 cattttttag tgaaatattt gataatgtaaaaggaataca agaaaattta ttaacaggta 3240 tgtttcgaag tatagaaacc agtatagtaatccaatcaga agaaaaggtt gatttgaatg 3300 aaaatgtggt tagttcgatt ttagataatatagaaaatat gaaagaaggt ttattaaata 3360 aattagaaaa tatttcaagt actgaaggtgttcaagaaac tgtaactgaa catgtagaac 3420 aaaatgtata tgtggatgtt gatgttcctgctatgaaaga tcaattttta ggaatattaa 3480 atgaggcagg agggttgaaa gaaatgttttttaatttgga agatgtattt aaaagtgaaa 3540 gtgatgtaat tactgtagaa gaaattaaggatgaaccggt tcaaaaagag gtagaaaaag 3600 aaactgttag tattattgaa gaaatggaagaaaatattgt agatgtatta gaggaagaaa 3660 aagaagattt aacagacaag atgatagatgcagtagaaga atccatagaa atatcttcag 3720 attctaaaga agaaactgaa tctattaaagataaagaaaa agatgtttca ctagttgttg 3780 aagaagttca agacaatgat atggatgaaagtgttgagaa agttttagaa ttgaaaaata 3840 tggaagagga gttaatgaag gatgctgttgaaataaatga cattactagc aaacttattg 3900 aagaaactca agagttaaat gaagtagaagcagatttaat aaaagatatg gaaaaattaa 3960 aagaattaga aaaagcatta tcagaagattctaaagaaat aatagatgca aaagatgata 4020 cattagaaaa agttattgaa gaggaacatgatataacgac gacgttggat gaagttgtag 4080 aattaaaaga tgtcgaagaa gacaagatcgaaaaagtatc tgatttaaaa gatcttgaag 4140 aagatatatt aaaagaagta aaagaaatcaaagaacttga aagtgaaatt ttagaagatt 4200 ataaagaatt aaaaactatt gaaacagatattttagaaga gaaaaaagaa atagaaaaag 4260 atcattttga aaaattcgaa gaagaagctgaagaaataaa agatcttgaa gcagatatat 4320 taaaagaagt atcttcatta gaagttgaagaagaaaaaaa attagaagaa gtacacgaat 4380 taaaagaaga ggtagaacat ataataagtggtgatgcgca tataaaaggt ttggaagaag 4440 atgatttaga agaagtagat gatttaaaaggaagtatatt agacatgtta aagggagata 4500 tggaattagg ggatatggat aaggaaagtttagaagatgt aacaacaaaa cttggagaaa 4560 gagttgaatc cttaaaagat gttttatctagtgcattagg catggatgaa gaacaaatga 4620 aaacaagaaa aaaagctcaa agacctaagttggaagaagt attattaaaa gaagaggtta 4680 aagaagaacc aaagaaaaaa ataacaaaaaagaaagtaag gtttgatatt aaggataagg 4740 aaccaaaaga tgaaatagta gaagttgaaatgaaagatga agatatagaa gaagatgtag 4800 aagaagatat agaagaagat atagaagaagataaagttga agatatagat gaagatatag 4860 atgaagatat aggtgaagac aaagatgaagttatagattt aatagtccaa aaagagaaac 4920 gcattgaaaa ggttaaagcg aaaaagaaaaaattagaaaa aaaagttgaa gaaggtgtta 4980 gtggtcttaa aaaacacgta gacgaagtaatgaaatatgt tcaaaaaatt gataaagaag 5040 ttgataaaga agtatctaaa gctttagaatcaaaaaatga tgttactaat gttttaaaac 5100 aaaatcaaga tttttttagt aaagttaaaaacttcgtaaa aaaatataaa gtatttgctg 5160 caccattcat atctgccgtt gcagcatttgcatcatatgt agttgggttc tttacatttt 5220 ctttattttc atcatgtgta acaatagcttcttcaactta cttattatca aaagttgaca 5280 aaactataaa taaaaataag gagagaccgttttattcatt tgtatttgat atctttaaga 5340 atttaaaaca ttatttacaa caaatgaaagaaaaatttag taaagaaaaa aataataatg 5400 taatagaagt aacaaacaaa gctgagaaaaaaggtaatgt acaggtaaca aataaaaccg 5460 agaaaacaac taaagttgat aaaaataataaagtaccgaa aaaaagaaga acgcaaaaat 5520 caaaataa 5528 2 1787 PRT K1Parasite Clone 2 Met Thr Asn Ser Asn Tyr Lys Ser Asn Asn Lys Thr Tyr AsnGlu Asn 1 5 10 15 Asn Asn Glu Gln Ile Thr Thr Ile Phe Asn Arg Thr AsnMet Asn Pro 20 25 30 Ile Lys Lys Cys His Met Arg Glu Lys Ile Asn Lys TyrPhe Phe Leu 35 40 45 Ile Lys Ile Leu Thr Cys Thr Ile Leu Ile Trp Ala ValGln Tyr Asp 50 55 60 Asn Asn Ser Asp Ile Asn Lys Ser Trp Lys Lys Asn ThrTyr Val Asp 65 70 75 80 Lys Lys Leu Asn Lys Leu Phe Asn Arg Ser Leu GlyGlu Ser Gln Val 85 90 95 Asn Gly Glu Leu Ala Ser Glu Glu Val Lys Glu LysIle Leu Asp Leu 100 105 110 Leu Glu Glu Gly Asn Thr Leu Thr Glu Ser ValAsp Asp Asn Lys Asn 115 120 125 Leu Glu Glu Ala Glu Asp Ile Lys Glu AsnIle Leu Leu Ser Asn Ile 130 135 140 Glu Glu Pro Lys Glu Asn Ile Ile AspAsn Leu Leu Asn Asn Ile Gly 145 150 155 160 Gln Asn Ser Glu Lys Gln GluSer Val Ser Glu Asn Val Gln Val Ser 165 170 175 Asp Glu Leu Phe Asn GluLeu Leu Asn Ser Val Asp Val Asn Gly Glu 180 185 190 Val Lys Glu Asn IleLeu Glu Glu Ser Gln Val Asn Asp Asp Ile Phe 195 200 205 Asn Ser Leu ValLys Ser Val Gln Gln Glu Gln Gln His Asn Val Glu 210 215 220 Glu Lys ValGlu Glu Ser Val Glu Glu Asn Asp Glu Glu Ser Val Glu 225 230 235 240 GluAsn Val Glu Glu Asn Val Glu Glu Asn Asp Asp Gly Ser Val Ala 245 250 255Ser Ser Val Glu Glu Ser Ile Ala Ser Ser Val Asp Glu Ser Ile Asp 260 265270 Ser Ser Ile Glu Glu Asn Val Ala Pro Thr Val Glu Glu Ile Val Ala 275280 285 Pro Ser Val Val Glu Ser Val Ala Pro Ser Val Glu Glu Ser Val Glu290 295 300 Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ser ValAla 305 310 315 320 Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu GluSer Val Ala 325 330 335 Glu Asn Val Glu Glu Ile Val Ala Pro Thr Val GluGlu Ile Val Ala 340 345 350 Pro Thr Val Glu Glu Ile Val Ala Pro Ser ValVal Glu Ser Val Ala 355 360 365 Pro Ser Val Glu Glu Ser Val Glu Glu AsnVal Glu Glu Ser Val Ala 370 375 380 Glu Asn Val Glu Glu Ser Val Ala GluAsn Val Glu Glu Ser Val Ala 385 390 395 400 Glu Asn Val Glu Glu Ser ValAla Glu Asn Val Glu Glu Ser Val Ala 405 410 415 Glu Asn Val Glu Glu IleVal Ala Pro Thr Val Glu Glu Ile Val Ala 420 425 430 Pro Thr Val Glu GluIle Val Ala Pro Ser Val Val Glu Ser Val Ala 435 440 445 Pro Ser Val GluGlu Ser Val Glu Glu Asn Val Glu Glu Ser Val Ala 450 455 460 Glu Asn ValGlu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala 465 470 475 480 GluAsn Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala 485 490 495Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala 500 505510 Glu Asn Val Glu Glu Ile Val Ala Pro Thr Val Glu Glu Ile Val Ala 515520 525 Pro Thr Val Glu Glu Ile Val Ala Pro Ser Val Val Glu Ser Val Ala530 535 540 Pro Ser Val Glu Glu Ser Val Glu Glu Asn Val Glu Glu Ser ValAla 545 550 555 560 Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu GluSer Val Ala 565 570 575 Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val GluGlu Ile Val Ala 580 585 590 Pro Thr Val Glu Glu Ile Val Ala Pro Thr ValGlu Glu Ile Val Ala 595 600 605 Pro Ser Val Val Glu Ser Val Ala Pro SerVal Glu Glu Ser Val Glu 610 615 620 Glu Asn Val Glu Glu Ser Val Ala GluAsn Val Glu Glu Ser Val Ala 625 630 635 640 Glu Asn Val Glu Glu Ser ValAla Glu Asn Val Glu Glu Ile Val Ala 645 650 655 Pro Thr Val Glu Glu IleVal Ala Pro Thr Val Glu Glu Ile Val Ala 660 665 670 Pro Ser Val Val GluSer Val Ala Pro Ser Val Glu Glu Ser Val Glu 675 680 685 Glu Asn Val GluGlu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala 690 695 700 Glu Asn ValGlu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala 705 710 715 720 GluAsn Val Glu Glu Ile Val Ala Pro Thr Val Glu Glu Ile Val Ala 725 730 735Pro Thr Val Glu Glu Ile Val Ala Pro Ser Val Val Glu Ser Val Ala 740 745750 Pro Ser Val Glu Glu Ser Val Glu Glu Asn Val Glu Glu Ser Val Ala 755760 765 Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala770 775 780 Glu Asn Val Glu Glu Ser Val Ala Pro Thr Val Glu Glu Ile ValAla 785 790 795 800 Pro Ser Val Glu Glu Ser Val Ala Pro Ser Val Glu GluSer Val Ala 805 810 815 Glu Asn Val Ala Thr Asn Leu Ser Asp Asn Leu LeuSer Asn Leu Leu 820 825 830 Gly Gly Ile Glu Thr Glu Glu Ile Lys Asp SerIle Leu Asn Glu Ile 835 840 845 Glu Glu Val Lys Glu Asn Val Val Thr ThrIle Leu Glu Asn Val Glu 850 855 860 Glu Thr Thr Ala Glu Ser Val Thr ThrPhe Ser Asn Ile Leu Glu Glu 865 870 875 880 Ile Gln Glu Asn Thr Ile ThrAsn Asp Thr Ile Glu Glu Lys Leu Glu 885 890 895 Glu Leu His Glu Asn ValLeu Ser Ala Ala Leu Glu Asn Thr Gln Ser 900 905 910 Glu Glu Glu Lys LysGlu Val Ile Asp Val Ile Glu Glu Val Lys Glu 915 920 925 Glu Val Ala ThrThr Leu Ile Glu Thr Val Glu Gln Ala Glu Glu Lys 930 935 940 Ser Ala AsnThr Ile Thr Glu Ile Phe Glu Asn Leu Glu Glu Asn Ala 945 950 955 960 ValGlu Ser Asn Glu Asn Val Ala Glu Asn Leu Glu Lys Leu Asn Glu 965 970 975Thr Val Phe Asn Thr Val Leu Asp Lys Val Glu Glu Thr Val Glu Ile 980 985990 Ser Gly Glu Ser Leu Glu Asn Asn Glu Met Asp Lys Ala Phe Phe Ser 9951000 1005 Glu Ile Phe Asp Asn Val Lys Gly Ile Gln Glu Asn Leu Leu ThrGly 1010 1015 1020 Met Phe Arg Ser Ile Glu Thr Ser Ile Val Ile Gln SerGlu Glu Lys 1025 1030 1035 1040 Val Asp Leu Asn Glu Asn Val Val Ser SerIle Leu Asp Asn Ile Glu 1045 1050 1055 Asn Met Lys Glu Gly Leu Leu AsnLys Leu Glu Asn Ile Ser Ser Thr 1060 1065 1070 Glu Gly Val Gln Glu ThrVal Thr Glu His Val Glu Gln Asn Val Tyr 1075 1080 1085 Val Asp Val AspVal Pro Ala Met Lys Asp Gln Phe Leu Gly Ile Leu 1090 1095 1100 Asn GluAla Gly Gly Leu Lys Glu Met Phe Phe Asn Leu Glu Asp Val 1105 1110 11151120 Phe Lys Ser Glu Ser Asp Val Ile Thr Val Glu Glu Ile Lys Asp Glu1125 1130 1135 Pro Val Gln Lys Glu Val Glu Lys Glu Thr Val Ser Ile IleGlu Glu 1140 1145 1150 Met Glu Glu Asn Ile Val Asp Val Leu Glu Glu GluLys Glu Asp Leu 1155 1160 1165 Thr Asp Lys Met Ile Asp Ala Val Glu GluSer Ile Glu Ile Ser Ser 1170 1175 1180 Asp Ser Lys Glu Glu Thr Glu SerIle Lys Asp Lys Glu Lys Asp Val 1185 1190 1195 1200 Ser Leu Val Val GluGlu Val Gln Asp Asn Asp Met Asp Glu Ser Val 1205 1210 1215 Glu Lys ValLeu Glu Leu Lys Asn Met Glu Glu Glu Leu Met Lys Asp 1220 1225 1230 AlaVal Glu Ile Asn Asp Ile Thr Ser Lys Leu Ile Glu Glu Thr Gln 1235 12401245 Glu Leu Asn Glu Val Glu Ala Asp Leu Ile Lys Asp Met Glu Lys Leu1250 1255 1260 Lys Glu Leu Glu Lys Ala Leu Ser Glu Asp Ser Lys Glu IleIle Asp 1265 1270 1275 1280 Ala Lys Asp Asp Thr Leu Glu Lys Val Ile GluGlu Glu His Asp Ile 1285 1290 1295 Thr Thr Thr Leu Asp Glu Val Val GluLeu Lys Asp Val Glu Glu Asp 1300 1305 1310 Lys Ile Glu Lys Val Ser AspLeu Lys Asp Leu Glu Glu Asp Ile Leu 1315 1320 1325 Lys Glu Val Lys GluIle Lys Glu Leu Glu Ser Glu Ile Leu Glu Asp 1330 1335 1340 Tyr Lys GluLeu Lys Thr Ile Glu Thr Asp Ile Leu Glu Glu Lys Lys 1345 1350 1355 1360Glu Ile Glu Lys Asp His Phe Glu Lys Phe Glu Glu Glu Ala Glu Glu 13651370 1375 Ile Lys Asp Leu Glu Ala Asp Ile Leu Lys Glu Val Ser Ser LeuGlu 1380 1385 1390 Val Glu Glu Glu Lys Lys Leu Glu Glu Val His Glu LeuLys Glu Glu 1395 1400 1405 Val Glu His Ile Ile Ser Gly Asp Ala His IleLys Gly Leu Glu Glu 1410 1415 1420 Asp Asp Leu Glu Glu Val Asp Asp LeuLys Gly Ser Ile Leu Asp Met 1425 1430 1435 1440 Leu Lys Gly Asp Met GluLeu Gly Asp Met Asp Lys Glu Ser Leu Glu 1445 1450 1455 Asp Val Thr ThrLys Leu Gly Glu Arg Val Glu Ser Leu Lys Asp Val 1460 1465 1470 Leu SerSer Ala Leu Gly Met Asp Glu Glu Gln Met Lys Thr Arg Lys 1475 1480 1485Lys Ala Gln Arg Pro Lys Leu Glu Glu Val Leu Leu Lys Glu Glu Val 14901495 1500 Lys Glu Glu Pro Lys Lys Lys Ile Thr Lys Lys Lys Val Arg PheAsp 1505 1510 1515 1520 Ile Lys Asp Lys Glu Pro Lys Asp Glu Ile Val GluVal Glu Met Lys 1525 1530 1535 Asp Glu Asp Ile Glu Glu Asp Val Glu GluAsp Ile Glu Glu Asp Ile 1540 1545 1550 Glu Glu Asp Lys Val Glu Asp IleAsp Glu Asp Ile Asp Glu Asp Ile 1555 1560 1565 Gly Glu Asp Lys Asp GluVal Ile Asp Leu Ile Val Gln Lys Glu Lys 1570 1575 1580 Arg Ile Glu LysVal Lys Ala Lys Lys Lys Lys Leu Glu Lys Lys Val 1585 1590 1595 1600 GluGlu Gly Val Ser Gly Leu Lys Lys His Val Asp Glu Val Met Lys 1605 16101615 Tyr Val Gln Lys Ile Asp Lys Glu Val Asp Lys Glu Val Ser Lys Ala1620 1625 1630 Leu Glu Ser Lys Asn Asp Val Thr Asn Val Leu Lys Gln AsnGln Asp 1635 1640 1645 Phe Phe Ser Lys Val Lys Asn Phe Val Lys Lys TyrLys Val Phe Ala 1650 1655 1660 Ala Pro Phe Ile Ser Ala Val Ala Ala PheAla Ser Tyr Val Val Gly 1665 1670 1675 1680 Phe Phe Thr Phe Ser Leu PheSer Ser Cys Val Thr Ile Ala Ser Ser 1685 1690 1695 Thr Tyr Leu Leu SerLys Val Asp Lys Thr Ile Asn Lys Asn Lys Glu 1700 1705 1710 Arg Pro PheTyr Ser Phe Val Phe Asp Ile Phe Lys Asn Leu Lys His 1715 1720 1725 TyrLeu Gln Gln Met Lys Glu Lys Phe Ser Lys Glu Lys Asn Asn Asn 1730 17351740 Val Ile Glu Val Thr Asn Lys Ala Glu Lys Lys Gly Asn Val Gln Val1745 1750 1755 1760 Thr Asn Lys Thr Glu Lys Thr Thr Lys Val Asp Lys AsnAsn Lys Val 1765 1770 1775 Pro Lys Lys Arg Arg Thr Gln Lys Ser Lys Glx1780 1785 3 1712 DNA T9/96 Parasite Clone 3 agtgatgaac tttttaatgaattattaaat agtgtagatg ttaatggaga agtaaaagaa 60 aatattttgg aggaaagtcaagttaatgac gatattttta atagtttagt aaaaagtgtt 120 caacaagaac aacaacacaatgttgaagaa aaagttgaag aaagtgtaga agaaaatgac 180 gaagaaagtg tagaagaaaatgtagaagaa aatgtagaag aaaatgacga cggaagtgta 240 gcctcaagtg ttgaagaaagtatagcttca agtgttgatg aaagtataga ttcaagtatt 300 gaagaaaatg tagctccaactgttgaagaa atcgtagctc caactgttga agaaattgta 360 gctccaagtg ttgtagaaagtgtggctcca agtgttgaag aaagtgtagc tccaagtgtt 420 gaagaaagtg tagctgaaaatgttgaagaa agtgtagctg aaaatgttga agaaatcgta 480 gctccaagtg ttgaagaaagtgtagctgaa aatgttgaag aaagtgtagc tgaaaatgtt 540 gaagaaagtg tagctgaaaatgttgaagaa agtgtagctg aaaatgttga agaaagtgta 600 gctgaaaatg ttgaagaaatcgtagctcca actgttgaag aaagtgtagc tccaactgtt 660 gaagaaattg tagctccaactgttgaagaa agtgtagctc caactgttga agaaattgta 720 gttccaagtg ttgaagaaagtgtagctcca agtgttgaag aaagtgtagc tgaaaatgtt 780 gaagaaagtg tagctgaaaatgttgaagaa agtgtagctg aaaatgttga agaaagtgta 840 gctgaaaatg ttgaagaaagtgtagctgaa aatgttgaag aaatcgtagc tccaagtgtt 900 gaagaaatcg tagctccaactgttgaagaa agtgttgctg aaaacgttgc aacaaattta 960 tcagacaatc ttttaagtaatttattaggt ggtatcgaaa ctgaggaaat aaaggacagt 1020 atattaaatg agatagaagaagtaaaagaa aatgtagtca ccacaatact agaaaaagta 1080 gaagaaacta cagctgaaagtgtaactact tttagtaata tattagagga gatacaagaa 1140 aatactatta ctaatgatactatagaggaa aaattagaag aactccacga aaatgtatta 1200 agtgccgctt tagaaaatacccaaagtgaa gaggaaaaga aagaagtaat agatgtaatt 1260 gaagaagtaa aagaagaggtcgctaccact ttaatagaaa ctgtggaaca ggcagaagaa 1320 gagagcgaaa gtacaattacggaaatattt gaaaatttag aagaaaatgc agtagaaagt 1380 aatgaaaaag ttgcagagaatttagagaaa ttaaacgaaa ctgtatttaa tactgtatta 1440 gataaagtag aggaaacagtagaaattagc ggagaaagtt tagaaaacaa tgaaatggat 1500 aaagcatttt ttagtgaaatatttgataat gtaaaaggaa tacaagaaaa tttattaaca 1560 ggtatgtttc gaagtatagaaaccagtata gtaatccaat cagaagaaaa ggttgatttg 1620 aatgaaaatg tggttagttcgattttagat aatatagaaa atatgaaaga aggtttatta 1680 aataaattag aaaatatttcaagtactgaa gg 1712 4 570 PRT T9/96 Parasite Clone 4 Ser Asp Glu Leu PheAsn Glu Leu Leu Asn Ser Val Asp Val Asn Gly 1 5 10 15 Glu Val Lys GluAsn Ile Leu Glu Glu Ser Gln Val Asn Asp Asp Ile 20 25 30 Phe Asn Ser LeuVal Lys Ser Val Gln Gln Glu Gln Gln His Asn Val 35 40 45 Glu Glu Lys ValGlu Glu Ser Val Glu Glu Asn Asp Glu Glu Ser Val 50 55 60 Glu Glu Asn ValGlu Glu Asn Val Glu Glu Asn Asp Asp Gly Ser Val 65 70 75 80 Ala Ser SerVal Glu Glu Ser Ile Ala Ser Ser Val Asp Glu Ser Ile 85 90 95 Asp Ser SerIle Glu Glu Asn Val Ala Pro Thr Val Glu Glu Ile Val 100 105 110 Ala ProThr Val Glu Glu Ile Val Ala Pro Ser Val Val Glu Ser Val 115 120 125 AlaPro Ser Val Glu Glu Ser Val Ala Pro Ser Val Glu Glu Ser Val 130 135 140Ala Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ile Val 145 150155 160 Ala Pro Ser Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val165 170 175 Ala Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu Glu SerVal 180 185 190 Ala Glu Asn Val Glu Glu Ser Val Ala Glu Asn Val Glu GluIle Val 195 200 205 Ala Pro Thr Val Glu Glu Ser Val Ala Pro Thr Val GluGlu Ile Val 210 215 220 Ala Pro Thr Val Glu Glu Ser Val Ala Pro Thr ValGlu Glu Ile Val 225 230 235 240 Val Pro Ser Val Glu Glu Ser Val Ala ProSer Val Glu Glu Ser Val 245 250 255 Ala Glu Asn Val Glu Glu Ser Val AlaGlu Asn Val Glu Glu Ser Val 260 265 270 Ala Glu Asn Val Glu Glu Ser ValAla Glu Asn Val Glu Glu Ser Val 275 280 285 Ala Glu Asn Val Glu Glu IleVal Ala Pro Ser Val Glu Glu Ile Val 290 295 300 Ala Pro Thr Val Glu GluSer Val Ala Glu Asn Val Ala Thr Asn Leu 305 310 315 320 Ser Asp Asn LeuLeu Ser Asn Leu Leu Gly Gly Ile Glu Thr Glu Glu 325 330 335 Ile Lys AspSer Ile Leu Asn Glu Ile Glu Glu Val Lys Glu Asn Val 340 345 350 Val ThrThr Ile Leu Glu Lys Val Glu Glu Thr Thr Ala Glu Ser Val 355 360 365 ThrThr Phe Ser Asn Ile Leu Glu Glu Ile Gln Glu Asn Thr Ile Thr 370 375 380Asn Asp Thr Ile Glu Glu Lys Leu Glu Glu Leu His Glu Asn Val Leu 385 390395 400 Ser Ala Ala Leu Glu Asn Thr Gln Ser Glu Glu Glu Lys Lys Glu Val405 410 415 Ile Asp Val Ile Glu Glu Val Lys Glu Glu Val Ala Thr Thr LeuIle 420 425 430 Glu Thr Val Glu Gln Ala Glu Glu Glu Ser Glu Ser Thr IleThr Glu 435 440 445 Ile Phe Glu Asn Leu Glu Glu Asn Ala Val Glu Ser AsnGlu Lys Val 450 455 460 Ala Glu Asn Leu Glu Lys Leu Asn Glu Thr Val PheAsn Thr Val Leu 465 470 475 480 Asp Lys Val Glu Glu Thr Val Glu Ile SerGly Glu Ser Leu Glu Asn 485 490 495 Asn Glu Met Asp Lys Ala Phe Phe SerGlu Ile Phe Asp Asn Val Lys 500 505 510 Gly Ile Gln Glu Asn Leu Leu ThrGly Met Phe Arg Ser Ile Glu Thr 515 520 525 Ser Ile Val Ile Gln Ser GluGlu Lys Val Asp Leu Asn Glu Asn Val 530 535 540 Val Ser Ser Ile Leu AspAsn Ile Glu Asn Met Lys Glu Gly Leu Leu 545 550 555 560 Asn Lys Leu GluAsn Ile Ser Ser Thr Glu 565 570 5 20 PRT Artificial Sequence Syntheticpeptide 5 Leu Leu Ser Asn Ile Glu Glu Pro Lys Glu Asn Ile Ile Asp AsnLeu 1 5 10 15 Leu Asn Asn Ile 20 6 25 PRT Artificial Sequence Syntheticpeptide 6 Asp Glu Leu Phe Asn Glu Leu Leu Asn Ser Val Asp Val Asn GlyGlu 1 5 10 15 Val Lys Glu Asn Ile Leu Glu Glu Ser 20 25 7 26 PRTArtificial Sequence Synthetic peptide 7 Leu Glu Glu Ser Gln Val Asn AspAsp Ile Phe Asn Ser Leu Val Lys 1 5 10 15 Ser Val Gln Gln Glu Gln GlnHis Asn Val 20 25 8 28 PRT Artificial Sequence Synthetic peptide 8 ValGlu Ser Val Ala Pro Ser Val Glu Glu Ser Val Ala Pro Ser Val 1 5 10 15Glu Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val 20 25 9 123 PRTArtificial Sequence Synthetic peptide 9 Leu Ala Ser Glu Glu Val Lys GluLys Ile Leu Asp Leu Leu Glu Glu 1 5 10 15 Gly Asn Thr Leu Thr Glu SerVal Asp Asp Asn Lys Asn Leu Glu Glu 20 25 30 Ala Glu Asp Ile Lys Glu AsnIle Leu Leu Ser Asn Ile Glu Glu Pro 35 40 45 Lys Glu Asn Ile Ile Asp AsnLeu Leu Asn Asn Ile Gly Gln Asn Ser 50 55 60 Glu Lys Gln Glu Ser Val SerGlu Asn Val Gln Val Ser Asp Glu Leu 65 70 75 80 Phe Asn Glu Leu Leu AsnSer Val Asp Val Asn Gly Glu Val Lys Glu 85 90 95 Asn Ile Leu Glu Glu SerGln Val Asn Asp Asp Ile Phe Asn Ser Leu 100 105 110 Val Lys Ser Val GlnGln Glu Gln Gln His Asn 115 120 10 96 PRT Artificial Sequence Syntheticpeptide 10 Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala Glu Asn ValGlu 1 5 10 15 Glu Ile Val Ala Pro Thr Val Glu Glu Ile Val Ala Pro ThrVal Glu 20 25 30 Glu Ile Val Ala Pro Ser Val Val Glu Ser Val Ala Pro SerVal Glu 35 40 45 Glu Ser Val Glu Glu Asn Val Glu Glu Ser Val Ala Glu AsnVal Glu 50 55 60 Glu Ser Val Ala Glu Asn Val Glu Glu Ser Val Ala Glu AsnVal Glu 65 70 75 80 Glu Ser Val Ala Glu Asn Val Glu Glu Ile Val Ala ProThr Val Glu 85 90 95

1 A vaccine composition comprising a Th1-inducing adjuvant incombination with a protecting Liver Stage Antigen or immunologicalfragment thereof of a human malaria parasite with the proviso that whenthe immunological fragment is an immunological fragment of LSA-3, theTh1-inducing adjuvant is not Montanide. 2 A vaccine composition asclaimed in claim 1 wherein the human malaria parasite is Plasmodiumfalciparum. 3 A vaccine composition as claimed in claim 1 or claim 2 inwhich the Th1-inducing adjuvant comprises either (a) QS21, De-O-acylatedmonophosphoryl lipid A (3D-MPL) and an oil in water emulsion wherein theoil in water emulsion has the following composition: a metabolisableoil, such a squalene, alpha tocopherol and tween 80; or (b) a vesicularadjuvant formulation comprising cholesterol, a saponin and optionally anLPS derivative. 4 A vaccine composition as claimed in claim 1 or 2 orclaim 3 wherein said protecting Liver Stage Antigen is the Liver StageAntigen 3 (LSA-3) or immunological fragment thereof. 5 A vaccinecomposition according to any one of claims 1 to 4 comprising in additionat least one other protecting antigen or an immunological fragmentthereof, of a malaria parasite. 6 A vaccine composition as claimed inclaim 4 in which the other malaria antigen is selected from thefollowing group: a) a hybrid protein comprising substantially all theC-terminal portion of the CS protein, four or more tandem repeats of theimmunodominant region, and the surface antigen from hepatitis B virus(HBsAg), in particular RTS,S, or immunogenic derivatives includingfragments thereof; b) the TRAP protein of the T9/96 isolate ofPlasmodium falciparum and proteins having at least 80% homology theretoand immunogenic derivatives including fragments thereof; c) the MSP-1 ofPlasmodium falciparum or Plasmodium vivax and proteins having at least80% homology thereto and immunogenic derivatives including fragmentsthereof; and d) the MSP-3 of Plasmodium falciparum or Plasmodium vivaxand proteins having at least 70% homology with the C-terminal regionthereof, and immunogenic derivatives including fragments thereof. 7 Avaccine composition according to claims 1 to 6 capable of involving a Tcell response in a mammal to the antigen or antigenic composition 8 Avaccine composition according to claims 1 to 7 capable of stimulatinginterferon γ production. 9 A vaccine composition according to claims 1to 8, wherein the ratio of QS21:3D-MPL is from 1:10 to 10:1. 10 Avaccine composition according to claims 1 to 8, wherein the ratio ofQS21:3D-MPL is from 1:1 to 1:2.5. 11 A process to make a vaccinecomposition according to any one of claims 1 to 10 comprising admixingQS21, 3D-MPL and the oil in water emulsion as defined in claim 2 with aprotecting Liver Stage Antigen of a human malaria parasite. 12 A processaccording to claim 11 wherein the Liver Stage Antigen is LSA-3 ofPlasmodium falciparum or immunological fragment thereof. 13 Use of acomposition according to any one of claims 1 to 10 for the prophylaxisor treatment of malaria infections.